• Journal of Microbiology and Biotechnology
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• 제10권5호
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• pp.656-662
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• 2000

A high level of Staphylococcus haemolyticus L62 lipase was expressed in an Escherichia coli transformant. The expressed lipase activity in the cell-free extract was 70,800 U/l, which corresponded to 30% of the total cellular protein. Pre-mixing of the l62 lipase with some nonionic detergents enhanced its hydrolytic activity towards olive oil: Tween detergents activated the L62 lipase by 3 fold. Gel filtration chromatography of the Tween-80-L62 lipase mixture demonstrated a polymerized complex (∼180 kDa) formed exclusively between Tween-80 and the L62 lipase. The lipase enzyme in the complex showed a higher specific activity towards most triacylglycerols than the intact L62 lipase. The activity enhancement towards each substrate was quite different depending on the acyl chain length; the activity towards tributyrin, trilinolein, and trilinolenin was much more enhanced than the towards the medium and the long-chain saturated triglycerides.

• Asian-Australasian Journal of Animal Sciences
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• 제29권10호
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• pp.1424-1431
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• 2016

The objective of this study was to evaluate the effect of diets containing different amounts of wheat, as a partial or whole substitute for corn, on digestibility, digestive enzyme activities, serum metabolite contents and ruminal fermentation in beef cattle. Four Limousin${\times}$LuXi crossbred cattle with a body weight ($400{\pm}10kg$), fitted with permanent ruminal, proximal duodenal and terminal ileal cannulas, were used in a $4{\times}4$ Latin square design with four treatments: Control (100% corn), 33% wheat (33% substitution for corn), 67% wheat (67% substitution for corn), and 100% wheat (100% substitution for corn) on a dry matter basis. The results showed that replacing corn with increasing amounts of wheat increased the apparent digestibility values of dry matter, organic matter, and crude protein (p<0.05). While the apparent digestibility of acid detergent fiber and neutral detergent fiber were lower with increasing amounts of wheat. Digestive enzyme activities of lipase, protease and amylase in the duodenum were higher with increasing wheat amounts (p<0.05), and showed similar results to those for the enzymes in the ileum except for amylase. Increased substitution of wheat for corn increased the serum alanine aminotransferase concentration (p<0.05). Ruminal pH was not different between those given only corn and those given 33% wheat. Increasing the substitution of wheat for corn increased the molar proportion of acetate and tended to increase the acetate-to-propionate ratio. Cattle fed 100% wheat tended to have the lowest ruminal $NH_3-N$ concentration compared with control (p<0.05), whereas no differences were observed among the cattle fed 33% and 67% wheat. These findings indicate that wheat can be effectively used to replace corn in moderate amounts to meet the energy and fiber requirements of beef cattle.

• KSBB Journal
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• 제13권5호
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• pp.577-584
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• 1998

A sensitive membrane strip assay for plasma lipoprotein cholesterol that can be performed without handling reagents has been investigated. We previously developed an assay system with immobilized enzymes (cholesterol esterase and cholesterol oxidase) on the surfaces of nitrocellulose membrane(1). In such a case, the amount of enzymes present on the membrane was limited by its surface area and, thus, the detection capability was relatively poor (> 50 mg/dL cholesterol). To overcome this problem, we devised a new system with non-immobilized enzymes by placing them within interstitial spaces of a celullose membrane pad in a dry state. Upon contact with sample medium, the enzymes were immediately dissolved and participated in the reactions with cholesterol in a liquid phase. We constructed a user-friendly system consisting of four membrane pads fro sample application, cholesterol decomposition, color development as signal, and medium absorption to invoke a continuous flow (sequential location from the bottom). A sample containing lipoproteins was added into the application pad by capillary action and transferred to the next pad for decomposition. The decomposition pad (namely, enzyme pad) contained a detergent (sodium cholate) for the destruction of lipoprotein particles, the two enzymes for cholesterol decomposition, and a chromogen (3,3'-diaminobenzidine). As a consequence of the enzyme reactions, hydrogen peroxide was produced, and then reacted in the presence of the chromogen with horseradish peroxidase immobilized on the signal generation pad. Finally, a colorimetric signal directly proportional to the cholesterol concentration was produced. The detection limit determined from this system under optimal conditions was at least 2 times lower than of the enzyme-immobilized system.

• Journal of Microbiology and Biotechnology
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• 제24권9호
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• pp.1260-1268
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• 2014

Screening of a gene library from Paenibacillus sp. PBS-2 generated in Escherichia coli led to the identification of a clone with lipolytic activity. Sequence analysis showed an open reading frame encoding a polypeptide of 378 amino acid residues with a predicted molecular mass of 42 kDa. The esterase displayed 69% and 42% identity with the putative ${\beta}$-lactamases from Paenibacillus sp. JDR-2 and Clostridium sp. BNL1100, respectively. The esterase contained a Ser-x-x-Lys motif that is conserved among all ${\beta}$-lactamases found to date. The protein PBS-2 was produced in both soluble and insoluble forms when E. coli cells harboring the gene were cultured at $18^{\circ}C$. The enzyme is a serine protein and was active against p-nitrophenyl esters of $C_2$, $C_4$, $C_8$, and $C_{10}$. The optimum pH and temperature for enzyme activity were pH 9.0 and $30^{\circ}C$, respectively. Relative activity of 55% remained at up to $5^{\circ}C$ with an activation energy of 5.84 kcal/mol, which indicates that the enzyme is cold-adapted. Enzyme activity was inhibited by $Cd^{2+}$, $Cu^{2+}$, and $Hg^{2+}$ ions. As expected for a serine esterase, activity was inhibited by phenylmethylsulfonyl fluoride. The enzyme was remarkably active and stable in the presence of commercial detergents and organic solvents. This cold-adapted esterase has potential as a biocatalyst and detergent additive for use at low temperatures.

• 미생물학회지
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• 제54권1호
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• pp.60-68
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• 2018

남극 해양세균 Pseudoalteromonas arctica PAMC 21717로부터 저온활성 alkaline protease (Pro21717)를 부분정제하였다. Pro21717 효소 추출액은 skim milk를 포함하는 zymogram gel 상에서 약 37 kDa (낮은 활성)과 74 kDa (높은 활성) 위치에서 두 개의 뚜렷한 투명밴드(clear zone)를 형성하였다. 단백질 분해활성을 나타내는 두 개의 효소단백질은 동일한 N-말단 아미노산 서열을 가지고 있었으며, 하나의 유전자에서 발현된 미성숙 단백질(precursor)이 37 kDa 크기의 단백질분해효소로 성숙화과정을 거친 후 74 kDa 크기로 이량체화됨으로써 좀 더 높은 활성을 가지는 것으로 판단된다. Pro21717은 $0-40^{\circ}C$ (최고활성 온도 $40^{\circ}C$) 온도 범위에서 단백질분해활성을 나타내었고 pH 5.0-10.0 (최적 pH 9.0) 범위에서 효소활성을 유지하였다. 주목할만한 특성으로써, Pro21717은 $40^{\circ}C$에서의 최고 효소활성(100%) 대비, $0^{\circ}C$와 $10^{\circ}C$에서 각각 30%와 45%의 높은 저온활성을 나타내었다. 또한 다양한 합성 펩타이드류에 대해 분해활성을 나타내는 Pro21717은 $Cu^{2+}$에 의해 활성이 증가하였으며, 시판용 세탁세제(commercial detergent formulation)에 포함되어 있는 다양한 종류의 계면활성제, 화학성분, 금속이온에 의해 활성이 감소되지 않았다. 전반적으로 저온활성 Pro21717은 글로벌 상업용효소 생산회사 Novozymes이 시판하고 있는 중온성 효소 Subtilisin Carlsberg (trademark Alcalase)에 버금가는 유용한 효소학적 특성이 있는 동시에 상대적으로 더 높은 저온활성을 보여주고 있다. 위의 실험결과들은, Pro21717은 $15^{\circ}C$ 이하의 차가운 수돗물에서도 세척력을 유지하는 새로운 세탁세제 효소첨가제로서의 개발 가능성을 보여주고 있다.

• Asian-Australasian Journal of Animal Sciences
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• 제30권1호
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• pp.57-63
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• 2017

Objective: Two experiments were conducted to determine the effects of adding exogenous phytase and xylanase, individually or in combination, as well as pelleting on nutrient digestibility, available energy content of wheat and the performance of growing pigs fed wheat-based diets. Methods: In Experiment 1, forty-eight barrows with an initial body weight of $35.9{\pm}0.6kg$ were randomly assigned to a $2{\times}4$ factorial experiment with the main effects being feed form (pellet vs meal) and enzyme supplementation (none, 10,000 U/kg phytase, 4,000 U/kg xylanase or 10,000 U/kg phytase plus 4,000 U/kg xylanase). The basal diet contained 97.8% wheat. Pigs were placed in metabolic cages for a 7-d adaptation period followed by a 5-d total collection of feces and urine. Nutrient digestibility and available energy content were determined. Experiment 2 was conducted to evaluate the effects of pelleting and enzymes on performance of wheat for growing pigs. In this experiment, 180 growing pigs ($35.2{\pm}9.0kg\;BW$) were allocated to 1 of 6 treatments according to a $2{\times}3$ factorial treatment arrangement with the main effects being feed form (meal vs pellet) and enzyme supplementation (0, 2,500 or 5,000 U/kg xylanase). Results: In Experiment 1, there were no interactions between feed form and enzyme supplementation. Pelleting reduced the digestibility of acid detergent fiber (ADF) by 6.4 percentage units (p<0.01), increased the digestibility of energy by 0.6 percentage units (p<0.05), and tended to improve the digestibility of crude protein by 0.5 percentage units (p = 0.07) compared with diets in mash form. The addition of phytase improved the digestibility of phosphorus (p<0.01) and calcium (p<0.01) by 6.9 and 7.6 percentage units respectively compared with control group. Adding xylanase tended to increase the digestibility of crude protein by 1.0 percentage units (p = 0.09) and increased the digestibility of neutral detergent fiber (NDF) (p<0.01) compared with control group. Supplementation of the xylanase-phytase combination improved the digestibility of phosphorus (p<0.01) but impaired NDF digestibility (p<0.05) compared with adding xylanase alone. In Experiment 2, adding xylanase increased average daily gain (p<0.01) and linearly improved the feed:gain ratio (p<0.01) compared with control group. Conclusion: Pelleting improved energy digestibility but decreased ADF digestibility. Adding xylanase increased crude protein digestibility and pig performance. Phytase increased the apparent total tract digestibility of phosphorus and calcium. The combination of phytase-xylanase supplementation impaired the effects of xylanase on NDF digestibility.

• Asian-Australasian Journal of Animal Sciences
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• 제29권4호
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• pp.539-548
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• 2016

The objective of the present study was to investigate the influence of palm kernel meal (PKM) inclusion and exogenous enzyme supplementation on growth performance, nitrogen-corrected apparent metabolizable energy (AMEn), coefficient of apparent ileal digestibility (CAID) and total tract retention of nutrients in young broilers fed corn-based diets. Four inclusion levels of PKM (no PKM [PKM0], 8% [PKM8], 16% [PKM16], and 24% [PKM24]) and two enzyme additions were evaluated in a $4{\times}2$ factorial arrangement of treatments. A total of 384, one-d-old male broilers (Ross 308) were individually weighed and allocated to 48 cages (eight broilers/cage), and cages were randomly assigned to eight dietary treatments. Results indicated that the inclusion of 8% and 16% PKM increased (p<0.05) the weight gain compared to the PKM0 diet. Birds fed the PKM8 diets had the highest (p<0.05) feed intake. Weight gain and feed intake were severely reduced (p<0.05) by feeding the PKM24 diet. Enzyme supplementation increased weight gain (p<0.05), independent of PKM inclusion level. In PKM0 and PKM8 diets, enzyme addition significantly (p<0.05) lowered feed conversion ratio (FCR); whereas enzyme addition had no effect on FCR of birds fed PKM16 and PKM24 diets. In PKM0 and PKM16 diets, enzyme addition significantly (p<0.05) increased CAID of nitrogen and energy but had no effect in the PKM8 and PKM24 diets. Inclusion of PKM into the basal diet, irrespective of inclusion level, enhanced (p<0.05) starch and fat digestibility. Inclusion of PKM at 16% and 24% resulted in similar CAID of neutral detergent fiber (NDF) but higher (p<0.05) than that of the PKM0 and PKM8 diets. Enzyme addition, regardless of the level of PKM inclusion, significantly (p<0.05) increased CAID of NDF. There was a significant (p<0.05) decrease in AMEn with PKM inclusion of 24%. The present data suggest that inclusion of PKM in broiler diets could be optimized if PKM-containing diets are formulated based on digestible amino acid contents and supplemented with exogenous enzymes. If amino acid digestibility and AME of PKM considered in the formulation, it can be included in broiler diets up to 16% with no deleterious effects on growth performance.

• Journal of Microbiology and Biotechnology
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• 제29권7호
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• pp.1043-1052
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• 2019

Active lipase-producing bacterium Burkholderia gladioli Bps-1 was rapidly isolated using a modified trypan blue and tetracycline, ampicillin plate. The electro-phoretically pure enzyme was obtained by purification using ethanol precipitation, ion-exchange chromatography, and gel filtration chromatography. The molecular weight was 34.6 kDa and the specific activity was determined to be 443.9 U/mg. The purified lipase showed the highest activity after hydrolysis with $p-NPC_{16}$ at a pH of 8.5 and $50^{\circ}C$, and the $K_m$, $k_{cat}$, and $k_{cat}/K_m$ values were 1.05 mM, $292.95s^{-1}$ and $279s^{-1}mM^{-1}$, respectively. The lipase was highly stable at $7.5{\leq}pH{\leq}10.0$. $K^+$ and $Na^+$ exerted activation effects on the lipase which had favorable tolerance to short-chain alcohols with its residual enzyme activity being 110% after being maintained in 30% ethanol for 1 h. The results demonstrated that the lipase produced by the strain B. gladioli Bps-1 has high enzyme activity and is an alkaline lipase. The lipase has promising chemical properties for a range of applications in the food-processing and detergent industries, and has particularly high potential for use in the manufacture of biodiesel.

• 한국의류학회지
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• 제22권2호
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• pp.224-232
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• 1998

Protease is mixtured in detergent to remove protein-soil easily. It must not act on the any fiber except protein-soil during laundry. So the purpose of this study is to investigate how protease is affect the fiber, particulary the protein-fiber. For this purpose, silk, wool and nylon are selected as samples, and the extent of the damage was estimated as tensile strength and surface condition (that is fibrillation). The results are as follows. The tensile strength of fiber treated with protease were lowered at enzyme concentration 0.1%, temperature 4$0^{\circ}C$ , and, as washing time was longer, it was lowered more. And it was showed that the surface of fibers were fiblliated by protease during washing. From this results, it was found that protease damaged protein-fiber. The damage of silk was the largest of all, and wool was less damaged than silk, because it has the scale (cuticle) on the outside. Additionary, an influence of surfactant on damage of fiber was little about three fibers, but, the fibers were damaged more by the binary nonionic-surfactant and protease mixture than by protease only.

• Asian-Australasian Journal of Animal Sciences
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• 제12권7호
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• pp.1063-1069
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• 1999

Wheat straw samples (3-4 cm) were sprayed with solutions of urea (U) alone or with a dry addition of garden soil (GS), midden soil (MS), soya bean meal (SM) or jack bean meal (JM) as crude urease sources and with a pure urease (UR) enzyme. Each of the urease sources was included at two levels: 30 and 60 g/kg except pure urease, which was added at a level of 2.5 & 5.0 g/kg treated straw dry matter. Untreated straw without urease source was used as a control. After treatment, samples were sealed in polythene bags and stored for 2, 7, 14, 21 and 35 days at $19{^{\circ}C}$. The urease sources, their levels and treatment time produced significant effects on ammonia production (p<0.01). The addition of urease offered more flexibility in hydrolyzing urea in the shortest possible time. Incorporation of soya bean and jack bean meal was effective in reducing the modified acid detergent fiber (MADF) content of straw and the same time increasing organic matter (OM) digestibility. Overall effect, addition of soya bean to urea at a ratio of 1:1 appeared to be the most satisfactory urease source for the treatment of urea and wheat straw.